Chemical Processes

Technical consistency (i.e. reproducibility and linearity) of the 3AA method was compared to two conventional chromatographic separations used for small molecule and amino acid analyses. Pancreatic cancer cell extracts (biological triplicates) either undiluted or diluted five-fold, were analyzed using (i) the 3AA method, as well as (ii) a 15 min gradient on a Kinetex HILIC column (150 × 2.1 mm i.d., 1.7 μm particle size – Phenomenex, Torrance, CA, USA) and (iii) a 23 min gradient on an Acquity UHPLC BEH Amide Column (2.1×100 mm, 1.7μm – Waters, Milford, MA, USA). In (ii), samples were analyzed on the Kinetex HILIC at 350 μl/min (mobile phases: A: ACN; B: 18 mΩ H₂O, 20 mM (NH₄)₂CO₃, 0.1% NH₄OH; gradient: 1.5 min hold at 5% B; 5-60% B in 8.5 min; 60-95% B in 0.5 min at 0.5 ml/min; 95% hold for 2 min; 95-5% B in 0.5 min; 5 hold for 2 min; column temperature: 25°C). In (iii), samples were analyzed on the Acquity UHPLC BEH Amide Column (2.1×100 mm, 1.7μm – Waters, Milford, MA, USA) at 500 μl/min (mobile phases: A: 18 mΩ H₂O, 20 mM (NH₄)₂CO₃ pH 4.00; B: acetonitrile; gradient: 3 min hold 85% B; 85-30% B in 9 min; 30-2% B in 3 min; 2-85% of B in 1 min; 85% equilibration for 7 min; column temperature: 60°C).
The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs.
MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012 ). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above.
Relative quantitation was performed by exporting integrated peak areas values into Excel (Microsoft, Redmond, CA, USA) for statistical analysis including T-Test and ANOVA (significance threshold for p-values < 0.05) and unsupervised Principal Component Analysis (PCA) (Pan et al. 2007 (link); Fonville et al. 2010 ), calculated through the MultiBase macro (freely available at www.NumericalDynamics.com).

The TOPAS-nBio extensions provide options for sub-cellular geometries, scoring, physics and chemistry. While most users will just need to adjust the values of the parameters used by the new classes, the classes can be modified with minimal coding requirements to adjust each aspect of a user’s simulation. Due to the modular nature of the extensions, users can select to only install the features they need for their simulations by downloading the necessary files and adding them to their TOPAS executable.
TOPAS-nBio facilitates and extends the use and configuration of the physical and chemical processes provided by Geant4-DNA (15 –18 ). The physical processes of Geant4-DNA, originally intended for radiation transport in liquid water, have recently included cross sections for DNA constituents (19 ), due to the availability of elastic and inelastic cross sections for these materials, which are also available in TOPAS-nBio.
The Geant4 toolkit provides users with basic 3D geometric shapes (solids), which include volumes such as boxes, ellipsoids, cylinders and spheres. All geometries in TOPAS-nBio are made up of either a single Geant4 solid, computer-aided-designed (CAD) solids or a combination of two or more of these solids. In some cases, unions of these solids are used (e.g., to create the double-helix DNA backbone from the union of spheres). The Geant4-DNA user community and others working on track-structure codes also work on new DNA or cell geometries and new DNA repair models. We actively collaborate with some of these groups to include the latest developments in our simulation framework. Several of the features presented here, e.g., interfaces to the DNAFabric code (20 –22 (link)) and DNA repair models (23 (link)–26 ), were developed as part of such collaborations.